Loteprednol etabonate nanoparticles in thermoreversible gels for enhanced therapeutics

ABSTRACT

The present invention provides a sustained drug delivery system for the treatment of age-related macular degeneration (AMD), comprising corticosteroid encapsulated nanoparticles incorporated into a thermoreversible hydrogel. The corticosteroid may be triamcinolone acetate (TA), dexamethasone, or loteprednol etabonate (LE). The proposed drug delivery system is nontoxic to ARPE-19 (retinal pigment epithelium) cells and significantly reduces VEGF (vascular endothelial growth factor) expression as compared to solutions of the coticosteroids. The present invention provides sustained delivery of the corticosteroid to the posterior segment of the eye, reducing the frequency of intraocular injections necessary to maintain therapeutic concentrations.

CROSS REFERENCE TO RELATED APPLICATION

This application claims priority to pending U.S. patent application Ser.No. 15/243,376, entitled “Nanoparticles in Thermoreversible Gels forEnhanced Therapeutics” filed on Aug. 22, 2016, which claims priority toU.S. Provisional Patent Application No. 62/207,997, entitled“Triamcinolone Acetonide Nanoparticles In Thermoreversible Gels ForEnhanced Therapeutics”, filed on Aug. 21, 2015, and U.S. ProvisionalPatent Application No. 62/208,187, entitled “Loteprednol EtabonateNanoparticles In Thermoreversible Gels For Enhanced Therapeutics”, filedon Aug. 21, 2015, the contents of which are herein incorporated byreference.

BACKGROUND OF THE INVENTION

Age-related macular degeneration (AMD) is the leading cause of visualloss in adults over the age of 50. Neovascular AMD is characterized bychoroidal neovascularization (CNV), the growth of abnormal blood vesselsunder the retina which can leak blood and fluid, causing deteriorationto the macula over time. Current treatment involves frequentintravitreal injections resulting in side effects, such as retinaldetachment and low patient compliance.

Accordingly, a method of sustained drug delivery for the treatment ofAMD is needed in the art that does not result in undesirable sideeffects or low patient compliance.

SUMMARY OF INVENTION

The present invention provides a sustained drug delivery system andmethod for the treatment of age-related macular degeneration. In variousembodiments, the drug delivery system of the present invention includescorticosteroid encapsulated nanoparticles incorporated into athermoreversible gel. The corticosteroid used may include triamcinoloneacetate (TA), loteprednol etabonate (LE) and dexamethasone. This is notintended to be limiting and other corticosteroids are within the scopeof the present invention.

In one embodiment of the present invention, triamcinolone acetonide (TA)is encapsulated by poly(ethylene glycol)-ylated (PEGylated)poly-(lactide-co-glycolide) (PLGA) nanoparticles (NPs) and incorporatedinto a PLGA-PEG-PLGA thermoreversible gel for the treatment of AMD. TheTA-loaded NPs are prepared using the nanoprecipitation method andcharacterized for basic parameters. The TA-loaded NPs show an averageparticle size of 208.00±1.00 nm and polydispersity index of 0.005±0.001.In a particular embodiment, the 20% (w/v) thermoreversible gel isprepared using the cold method. MTT cytotoxicity data show that the drugdelivery system was not cytotoxic on ARPE-19 cells as opposed to anequivalent concentration of TA alone. In vitro release analysisdemonstrates that free TA was completely released within 48 hours;whereas 94% of TA was released from drug delivery system after 7 days.The proposed delivery system for TA is effective for sustained releasetreatment of ocular dysfunction.

In another embodiment, the present invention provides a sustained drugdelivery system for the treatment of AMD which is comprised ofloteprednol etabonate, poly(ethylene glycol)-ylated (PEGylated)poly-(lactide-co-glycolide) (PLGA) nanoparticles (NPs), and aPLGA-PEG-PLGA thermoreversible gel. This embodiment of the presentinvention provides sustained delivery of loteprednol etabonate to theposterior segment of the eye, thereby reducing the frequency ofintraocular injections necessary to maintain therapeutic concentrations.In this particular embodiment of the present invention, the proposeddrug delivery system is characterized for drug release, cytotoxicitystudies and vascular endothelial growth factor (VEGF) suppressionefficacy studies using ARPE-19 cells. The nanoparticles show uniformsize distribution with mean size of 168.60±23.18 nm and exhibitsustained drug release. Additionally, the proposed drug delivery systemis noncytotoxic to ARPE-19 cells and significantly reduced VEGFexpression, as compared to loteprednol etabonate solution. These resultssuggest that the proposed drug delivery system comprised of loteprednoletabonate, poly(ethylene glycol)-ylated (PEGylated)poly-(lactide-co-glycolide) (PLGA) nanoparticles (NPs), and aPLGA-PEG-PLGA thermoreversible gel can be used for treating AMD, therebyreducing intravitreal administration frequency.

As such, the present invention provides sustained delivery oftriamcinolone acetonide and loteprednol etabonate to the posteriorsegment of the eye, thereby reducing the frequency of intraocularinjections necessary to maintain therapeutic concentrations.

BRIEF DESCRIPTION OF THE DRAWINGS

For a fuller understanding of the invention, reference should be made tothe following detailed description, taken in connection with theaccompanying drawings, in which:

FIG. 1 is a graphical illustration of the temperature-dependent phasetransitions in PLGA-PEG-PLGA thermoreversible gels based on w/vconcentration of gel in aqueous solution, in accordance with anembodiment of the present invention.

FIG. 2 is a graphical illustration of in vitro release data of the TA NPand TA NP gel as compared to equal concentrations of the TA drug.Samples were analyzed by UV spectroscopy (240 nm, λ_(max)) atpredetermined intervals over a 10-day period (n=3, mean value±SD), inaccordance with an embodiment of the present invention.

FIG. 3 is a graphical illustration of MTT cytotoxicity data in ARPE-19cells of equal treatments (10 μM) of the following: Blank NP, TA freedrug, TA NPs, TA drug in 20% w/v TR gel, and TA NPs in 20% w/v TR gel ascompared to untreated control cells, in accordance with an embodiment ofthe present invention.

FIG. 4 is a graphical illustration of time-dependent inhibition of VEGFsecretion in ARPE-19 cells through equal concentration treatments (100μM) of blank NPs, TAfree drug and TA NPs at 12 and 72 h, in accordancewith an embodiment of the present invention.

FIG. 5 is a graphical illustration of comparative suppression of VEGFsecretion in ARPE-19 cells through equal concentration treatments (10μM) of TA free drug, TA NPs, TA drug in 20% w/v TR gel, and TA NPs in20% w/v TR gel at 72 h, in accordance with an embodiment of the presentinvention.

FIG. 6 is a graphical illustration of in vitro release data of theloteprednol etabonate nanoparticles (LE NP) compared to LE NP gel, inaccordance with an embodiment of the present invention.

FIG. 7 is a graphical illustration of MTT cytotoxicity data in ARPE-19cells of increasing concentrations of loteprednol etabonate (LE) freedrug, LE NPs, and LE NPs in 20% w/v TR gel as compared to untreatedcontrol cells, in accordance with an embodiment of the presentinvention.

FIG. 8 is a graphical illustration of comparative suppression of VEGFsecretion in ARPE-19 cells through increasing concentrations (1, 10, μM)of loteprednol etabonate (LE) free drug, LE NPs, and LE NP gel at 12hours, in accordance with an embodiment of the present invention.

FIG. 9 is a graphical illustration of comparative suppression of VEGFsecretion in ARPE-19 cells through equal concentration treatments (10μM) of LE free drug, LE NPs, and LE NPs in 20% w/v TR gel at 72 hours,in accordance with an embodiment of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

AMD, a progressive, inflammatory eye disease, is one of the leadingcauses of blindness. It has been estimated that 36.8 million peoplesuffered from some sort of vision loss due to eye diseases in the UnitedStates in 2010. AMD affects the posterior segment of the eye by damagingretinal pigment epithelium (RPE) cells which leads to a loss of central,focused vision through the abnormal growth of blood vessels damaging themacula of the retina, the area responsible for fine vision. The presenceof intraocular debris may induce an inflammatory response which maycause further damage to the retina through the induction of a sustainedimmune response. Advanced AMD is characterized by choroidalneovascularization (CNV), the growth of abnormal blood vessels beneaththe RPE or between the RPE and retina, accompanied by fluid and bloodrupturing Bruch's membrane into the subretinal space and leading toretinal irregularities. Physical ocular barriers and routes of treatmentpose limitations to therapeutic intervention in treating AMD.

Anti-angiogenic therapy is useful in slowing the progression of AMD dueto the neovascularization characteristic of the disease. Vascularendothelial growth factor A (VEGF-A) is the most potent promoter ofangiogenesis and vascular permeability, and its role in the pathogenesisof neovascular AMD is well recognized. VEGF-A levels are elevated inhuman CNV, and its vitreous levels have been reported to be increasedwhen compared to healthy controls. VEGF is a potential pharmaceuticaltarget; elevated VEGF levels can increase inflammation via inducinginflammatory mediators like intercellular adhesion molecule-1 (ICAM-1)and subsequently lead to the breakdown of the blood-retinal barrier(BRB).

The anatomy of the eye is a challenging part of the body for drugdelivery. The posterior segment of the eye consists of the retina,vitreous and choroid. Topical treatment can easily access the anteriorportion of the eye; however, multiple physical barriers and clearancemechanisms prevent easy access to the posterior segment of the eye. Thetopical route is the most favored and convenient for drug delivery, butnasolacrimal drainage and systemic absorption result in a poor drugbioavailability. A model of transient diffusion has shown that less than5% of a lipophilic drug and 0.5% of a hydrophilic drug reach theanterior chamber. The bioavailability of the drug further decreasesacross the sclera, choroid and RPE. Drug permeability through the sclerais reduced with cationic and lipophilic solutes, and RPE have tightintercellular junctions to prevent the permeation of hydrophilicmolecules. Furthermore, the lymphatic system, blood vessels and activetransporters all work to clear drugs administered through transscleralroutes. Drug delivery through systemic administration requires highdoses to obtain a therapeutic concentration in the posterior segment ofthe eye due to the tight barriers in RPE. Intravitreal injectionscircumvent the physiological barriers and maintain therapeutic doseswithout damaging bystander tissue; however, frequent injections can leadto complications like retinal detachment, increase in ocular pressureand hemorrhage. Given the presence of these physiological barriers, thedevelopment of therapies that efficiently, deliver drugs and extend drugrelease to the posterior segment of the eye would be beneficial to theprogression of ocular disease treatment.

Although current therapies exist to slow the progression of AMD,alternative drug delivery systems (DDS) are needed to enhance thetherapeutic profiles of these drugs. Nanoparticles (NP) have beenstudied as drug carriers in ocular pharmaceuticals. They, can be madefrom biodegradable, polymeric materials in which drugs can be dissolved,entrapped or adsorbed. The major benefits of implementing NPs for oculardrug delivery are: (1) ease of administration via injection due to thesize of the particle, (2) smaller particles are well tolerated in theeye, (3) particles of the nanometer range have shown increasedsolubility, surface area and drug dissolution, (4) NPs can bemanipulated by polymer's weight and hydrophilicity to allow sustaineddrug release and (5) particles approximately 200 nm in size can belocalized in RPE cells. In addition, recent studies have shown thatafter intravitreal injections, a majority of NPs were localized at theRPE within 6 h and cytoplasmic concentrations of the NP remainedelevated for as long as 4 months.

Poly(lactide-co-glycolic acid) (PLGA) is an FDA-approved polymer thathas been studied due to its biocompatibility and toxicity. The rate ofdegradation can be manipulated by the polymer's molecular weight,hydrophilicity and ratio of lactide to glycolide to extend the releasetime of associated drugs. PLGA NPs have been shown to have controlleddrug release, low cytotoxicity and few side effects. Polyethylene glycol(PEG) has a slow clearance from the blood, allowing an increased drugrelease and reducing PLGA NP uptake by the reticulo-endothelial system(RES) when chemically conjugated to the PLGA vector as compared tonon-conjugated PLGA. PEGylated PLGA NPs of bevacizumab, an anti-VEGFantibody, have shown sustained release of the drug over 60 days.

Thermoreversible hydrogels (TR gels) are a subtype of in situ gels thatcan be administered as a solution and undergo gelation with specificstimuli. Specifically, TR gels are biodegradable, water soluble polymersthat undergo phase transitions upon temperature elevation. The gel canbe loaded with bioactive macromolecules and pharmacological agentsirrespective of their solubility properties. Localized and sustainedrelease can be achieved due to the gel's quick formation in vivo. Theirimproved bioavailability and ease of administration make them anattractive DDS. Studies have shown the synthesis of an ABA-type blockcopolymer, polyethylene glycol)-poly(serinol hexamethylene urethane), torelease bevacizumab. The in vitro drug release profile achieved a longertherapeutic window over 17 weeks. Such treatments could potentiallyreduce the frequency of intravitreal injections. An alternativeformulation includes Pluronic F 127 as the thermoreversible polymer andmethyl cellulose as a release controlling agent. This formulation hasbeen used to deliver nonsteroidal anti-inflammatory drugs forconjunctivitis as well as selective inhibitors for glaucoma.

Triamcinolone acetate (TA), a synthetically modified glucocorticoid, isutilized for its anti-inflammatory and immunomodulatory effects againsta number of diseases. TA is commonly used in conjunction with otherdrugs and acts by binding steroid receptors in cells and subsequentlyinducing or repressing target genes, thus leading to an inhibition ofinflammatory processes, such as edema and vascular permeability.

TA, and similar glucocorticoids, also act on vascular endothelial growthfactor (VEGF) by inhibiting its secretion and inhibiting cytokineproduction. Such drugs can also inhibit basic fibroblast growth factor(bFGF) along with decreasing the VEGF levels characteristic ofneovascularization. TA is known to inhibit laser-induced choroidalneovascularization in rats as well as improve visual acuity wheninjected intravitreally.

However, high doses of TA and similar steroids may lead to adverseeffects such as increased intraocular pressure and the formation ofcataracts. TA is clinically used intravitreally againstneovascularization in age-related macular degeneration (AMD).

In one embodiment, the present invention describes the preparation ofTA-encapsulated PLGA-PEG NPs incorporated into TR gel to improve thetherapeutic profile of TA in AMD. It is shown that the NP size isappropriate for intracellular uptake, the NPs and TR gel demonstrate asustained release of the drug over 10 days, the NP and TR gel vectorsare non-toxic in ARPE-19 cells, and the NP and TR gel DDS are able toreduce VEGF levels in ARPE-19 cells. These results suggest that theproposed DDS has the potential to significantly improve currenttherapies against AMD.

In an exemplary embodiment of the present invention,Poly(lactic-co-glycolic acid) (PLGA) conjugated with polyethylene glycol(PEG) (PEG-PLGA) (5050 DLG, mPEG 5000, 5 wt % PEG) was purchased fromLakeshore Biomaterials (Birmingham, Ala.). Triamcinolone acetonide waspurchased from Alfa Aesar (Ward Hill, Mass.).Poly(lactide-co-glycollide)-b-Poly(ethyIeneglycol)-b-Poly(lactide-co-glycolide)[PLGA-PEG-PLGA (Mn˜100:1,000:1,100 Da, 3:1 LA:GA) (25% PEG)]thermogelling polymer was purchased from Polyscitech (West Lafayette,Ind.). Phosphate-buffered saline (PBS) solution was purchased fromMediatech, Inc (Manassas, Va.). Thiazolyl blue tetrazolium bromide (MTTreagent), acetone and methanol were purchased from Sigma Aldrich (St.Louis, Mo.). All other chemicals used in this exemplary embodiment wereof analytical grade and were used without any further purification,unless specified.

In the exemplary embodiment, TA NPs were prepared using a previouslyreported nanoprecipitation method. Briefly, 22 mg of TA and 110 mg ofPEGylated PLGA were dissolved in 2 ml acetone (organic phase), and theresulting solution was added dropwise to 20 ml of deionized H2O (40° C.)spinning at 300 rpm overnight to allow the full evaporation of theorganic phase and the formation of the NP suspension. The NPs wereseparated by centrifugation at 3000 rpm for 10 min and resuspended infresh deionized H2O.

The NP parameters were studied by measuring the particle size andpolydispersity index (PDI). The effect of loading TA into the NPs onthese parameters was studied by, comparing TA NPs to blank NPs. Particlesize and PDI were measured using the Dynamic Pro plate reader (WyattTechnology Corporation, Santa Barbara, Calif.). The NP samples werediluted 1:5 in deionized water to fit instrument specifications.

Drug encapsulation efficiency was determined using a previously reportedmethod. 1 ml of the NP solution was centrifuged at 12 000 rpm for 5 min.The supernatant was removed and was replaced with 1 ml of methanol andstored at 4° C. overnight. The supernatant of the NP/methanol solutionwas diluted 1:5 times and measured by UV spectroscopy at a wavelength of240 nm (λ_(max)). The supernatant was analyzed and compared to a seriesof standard dilutions of TA in methanol (r²=0.99764). Encapsulationefficiency was determined using the following equation:

${\%\mspace{14mu}{Entrapment}{\mspace{11mu}\;}{efficiency}} = {\frac{{Actual}{\mspace{11mu}\;}{drug}\mspace{14mu}{amount}}{{Theoretical}\mspace{14mu}{drug}\mspace{14mu}{amount}} \times 100}$

TR gels were prepared using the cold method. To prepare 20% w/v gel, 100mg of PLGA-PEG-PLGA gel was solubilized in deionized H2O overnight at 4°C. Different percentages of the gel (10, 15, 20, 25 and 30% w/v) werescreened for the most optimal gelation temperature.

Gelation temperature was detected by visual inspection using apreviously published method. Aqueous solutions of the gel (10, 15, 20,25 and 30% wily) were prepared in distilled deionized H₂O, and 500 mL ofeach solution was heated from 10 to 60° C. At each 1° C. interval, thetubes were inverted to investigate flow properties. Solutions wereconsidered to be in the gel state if no flow was observed following tubeinversion.

The rate of TA release from the NPs was measured by adapting apreviously reported method. Dialysis membrane tubing, MWCO 10 000 Da.(Spectrum Laboratories, Rancho Dominguez, Calif.) was soaked indeionized 1120 overnight. 1 ml of the NP suspension was added to thedialysis membrane tubing after being briefly sonicated and was placedinto 100 ml of PBS covered and stirring at 100 rpm at 37° C. 1 mLsamples were removed at predetermined intervals over 10 days andreplaced with fresh PBS. Samples were analyzed using UV spectroscopy ata wavelength of 240 nm (λmax) and compared to standard dilutions of TAin PBS (r²=0.989) to determine the percentage of drug released over 10days.

The rate of TA release from the TR gel was measured by dispersing TA NPsin 200 ml of 20% w/v polymer solution and allowed to gel by placing inincubator at 37° C. for 5 min. After gelling, release was initiated byadding 2.5 ml PBS. 1 ml samples were removed at predetermined intervalsover 10 days and replaced with fresh PBS.

Human retinal pigment epithelium, ARPE-19, cells (ATCC # CRL-2302) weregrown in 1:1 Dulbecco's Modified Eagle's Medium and Ham's F12 Medium(Mediated, Inc., Manassas, Va.) with 10% fetal bovine serum and 100 Um′penicillin/streptomycin (Sigma Aldrich, St. Louis, Mo.). The cultureswere maintained in a humid environment at 5% CO₂ and 37° C.

The cytotoxicity of the DDS was assessed in ARPE-19 cells by the3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide salt (MTT)assay. Briefly, cells were seeded in 24-well plates at a density of5×104 cells/ml for 24 h to achieve confluency before treatment. Cellswere exposed to free TA, TA NPs, and TA NP-TR gel at varyingconcentrations (0, 1, 10, 100 μM) for 24 h after which the MTT assay wasperformed. Cell culture media were aspirated, and 500 ml of MTT reagentsolution (1 mg/ml) was added to each well. Cells were incubated at 37°C. and 5% CO₂ for 4 h, after which the MTT reagent solution wasaspirated and 1 mL of DMSO was added to each well. Plates were allowedto shake gently for 10 min before being read by Synergy H4 plate reader(Biotek Industries, Inc., Winooski, Vt.) at absorbance of 570 nm.

ARPE-19 cells were seeded onto 24-well plates at 5×104 cells/ml andallowed to grow until confluence. On the day of study, cell culturemedia were replaced with 1% FBS experimental media and allowed to remainin quiescence for 12 h. The cellular monolayers were incubated withvarying treatment concentrations of free TA solution (1, 10 and 100 μM),TA NPs (10 and 100 μM) and TA NP-TR gel (10 and 100 μM). Culture mediawere collected at 12 and 72 h. Secreted VEGF in collected culture mediawas quantified by the ELISA method (Human VEGFA ELISA kit, ThermoScientific, Waltham, Mass.). The cell protein content was assayed usingthe BCA protein assay kit after lysing the cells, and VEGF secretion wasnormalized to total protein. Samples were read using the Synergy H4plate reader (Biotek Industries, Inc., Winooski, Vt.) with absorbance at450 nm minus absorbance at 550 nm.

Statistical analyses were performed using Graph Pad Prism (Graph PadSoftware, Inc., San Diego, Calif.), Comparisons of the effect of free TAsolution, TA NPs and TA NP-TR gels on cell viability and VEGF expressionwere assessed using paired t tests with a significance level (p) of0.05, All experiments were carried out in triplicates (n=3) and shown asmean values±SD.

Dynamic Pro plate reader was used to determine the size and PDI of theblank and TA-loaded NPs. The TA NPs were larger in size than the blankNPs. PDI data revealed that all NP formulations had a narrow averagesize distribution (Table 1).

TABLE 1 NP characterization data carried out in triplicates (n = 3) andrepresented as the mean yalue ± SD. NP type Particle size (nm) PDI BlankNP 125.10 ± 43.89 0.014 ± 0.004 TA NP 208.00 ± 1.00 0.005 ± 0.001

UV spectroscopy was used to determine the encapsulation efficiency of TAby comparing the absorbance in methanol to standard dilutions of TA inmethanol (r²=0.99764) at 240 nm (λmax). It was found that 42.4±0.09% ofthe TA added during formulation was taken up by the NPs.

Phase transition studies revealed that the 15% w/v aqueous solution ofthe polymer converts to the gel phase at 40° C. and remains in a gelstate until 47° C. The gel phase is broader with higher % w/v solutions.The 20% w/v gel solution demonstrated an optimal transition withinphysiological conditions and was employed for future studies. FIG. 1shows the temperature at which each gel formulation transitioned fromthe solution state to gel state.

The release of TA from NPs and TR gel was investigated in 100 ml of PBSand 37° C. FIG. 2 shows the cumulative release profile of the TA NPformulation and TA NP Gel as compared to equal concentrations of thefree TA solution. At 48 h; free TA was fully released while the TA NPsreleased 48.9±7.0% of encapsulated drug and the TA NP Gel released23.24±6.7% of drug. At 168 h, the cumulative release of TA from the NPformulations was 94.17±20.8%. There is a lack of initial burst releaseof drug from the NPs. TR Gel had released 31.49±5.1% of drug at 10 days.

The cytotoxicity of the DDS was investigated by NM assay in the ARPE-19cells to determine any possible harm related to its use. FIG. 3 comparesthe cytotoxicity data of the free TA with equal concentrations (10 μM)of the different TA DDS's described in the present exemplary embodiment:the blank NPs, TA NPs, the TA in 20% w/v TR gel, and the TA NP in 20%w/v TR gel. The free TA induced significant cell death in ARPE-19 cellsas compared to the untreated control cells. All other treatments werenot significantly cytotoxic in ARPE-19 cells.

The effect of the different TA NP formulations on VEGF secretion wasstudied in ARPE-19 cells. Cells were treated with differentconcentrations of free TA, TA NPs, TA in 20% w/v TR gel and TA NP in 20%w/v TR gel, for 12 and 72 h. MG. 4 compares the time-dependentsuppression of VEGF expression among blank NPs, free TA and TA NPs (100μM) at 12 and 72 h. The blank NPs did not reduce VEGF expressionthroughout the time periods tested. The free TA significantly reducedVEGF secretion in 12 h, but TA NPs were not able to do it; however, at72 h; free TA was unable to significantly reduce VEGF secretion but TANPs were able to reduce VEGF secretion. FIG. 5 compares the effect ofdifferent DDS's (10 μM) on VEGF expression at 72 h: free TA, TA NPs, TAin 20% w/v TR gel and TA NPs in 20% w/v TR gel. The TA NPs, TA in 20%w/v TR gel and TA NPs in 20% w/v TR gel all significantly reduced VEGFexpression after 72 h, but the free TA alone had no significant effect.

The emulsion solvent evaporation process was used in the formulation ofthe polymeric TA NP suspension. Multiple formulations revealedconsistent reproducibility with respect to particle size andencapsulation efficiency (data not shown). Particle size data showedthat the average size of the NPs increases with drug loaded into the NPsas compared to the blank NPs (180-208 nm). Each of the NP formulationsexhibited a unimodal size distribution, suggesting a Gaussiandistribution with respect to NP size in each formulation. Particle size,PDI and encapsulation efficiency data were corroborated with previouslypublished studies.

The release of TA from the TA NPs and TR gel was measured in 37° C. PBSover 10 days as compared to the release of the free TA. TA remainsstable in solution as demonstrated by release data previously reported.The free TA was completely released from the dialysis membrane in thefirst 48 h; however, only approximately 48.9±7.0% of the drug wasreleased from each of the NP formulations in as much time. Almost all ofthe drug was released from the NPs by the end of the 10-day period. Thelack of initial burst release suggests that the drug was wellencapsulated by the PLGA NP during formulation and none of the drugexists on the surface of the NP. The NP formulation exhibited asustained release over the 10-day period tested. The release ofentrapped drug from a polymer matrix is believed to occur in two stages:the early stage of drug release occurs through diffusion in the polymermatrix, while the latter phases occur through a combination of diffusionand polymer degradation. 49±5.1% of the drug was released from the TRgel at 10 days. Similar results are reported in the literature where TRgel based on PLGA and PEG were able to deliver a 45-kDa protein acrossthe sclera to the retina for up to 14 days. The gelation temperatures ofdifferent % w/v formulations of the PLGA-PEG-PLGA TR gel wereinvestigated, it was found that an increasing % w/v of the TR gelsolution broadened the temperature range over which the TR gel changesphases from solution to gel to solution (sol-gel-sol). The temperaturesover which the 20% w/v TR gel phase transitioned from sol-gel-sol werebest in regards to physiological conditions. The 20% w/v TR geltransitions from solution to gel at approximately 32° C., which is theoptimum temperature for its easy intraocular injection and phasetransition into the gel phase at physiological temperatures. Theincorporation of a 10 μM treatment of TA NP formulation into the 20% w/vgel did not produce any cytotoxic effects on ARPE-19 suggesting its safeuse in in vivo settings.

NP cytotoxicity studies in ARPE-19 cells showed that the PLGA NP vectoris not cytotoxic although treatments with 10 μM free TA are cytotoxicunder similar conditions. It was also found that not only is the PLGAvector safe for use in ARPE-19 cells but also that the TA NP formulationand TR gel DDS (with incorporated TA NPs) are not significantlycytotoxic. Concentrations of TA needed to inhibit VEGF secretion havebeen previously reported. Consistent with those values, the results showthat the TA NPs and TR DDS formulations are able to significantly reduceVEGF expression in ARPE-19 cells at 10 and 100 μM over 72 h moreeffectively than the free TA at equal concentrations. The free TA isable to significantly reduce VEGF expression at the 100-μM concentrationin 12 h and similar concentrations take the TA NPs 72 h to reduce VEGFexpression, which may be due to the extended release properties of theTA NPs.

As such, in this embodiment of the present invention, a novel DDS isprovided for the amelioration of the CNV exhibited in cases of AMD. Inthis embodiment, the DDS comprises TA encapsulated NPs that areincorporated into 20% w/v TR gels. The TA NPs are spherical and theshape and sizes are compatible in the cells. The NPs exhibited anextended and sustained release of TA as is evident from in vitro releasedata. Furthermore, the TA NPs and NP-incorporated 20% w/v TR gel did notexhibit cytotoxicity in ARPE-19 cells in concentrations that are shownto significantly reduce ARPE-19 cell viability by free TA alone underthe same conditions. The TA NP and NP-incorporated 20% w/v TR gel wereable to significantly reduce the expression of VEGF in ARPE-19 cellsover 72 h at concentrations less than those required by free TA alone(10 μM TA-loaded DDS versus 100 μM free TA). The biocompatibility andtherapeutic potential of the proposed DDS makes it an effective modelfor the treatment of AMD.

Loteprednol etabonate (LE), a derivative of prednisolone was the firstretrometabolically designed steroid. It is a siteactive corticosteroiddesigned to transform into an inactive metabolite after exerting itstherapeutic effect. It contains an ester group at the carbon 20position, instead of a ketone. Compared to prednisolone acetate, it isless likely to increase ocular pressure and does not lead to cataractformation. Therefore, it is commonly, prepared as a suspension and usedtopically for anterior segment disorders such as allergy conjunctivitis,anterior uveitis, and postoperative inflammation.

The present invention describes a drug delivery system consisting ofloteprednol etabonate encapsulated PLGA-PEG NPs incorporated into TR gelto improve the therapeutic profile of loteprednol etabonate in AMD. Theproposed DDS has the potential to significantly improve currenttherapies against CNV.

In a particular embodiment, Poly(lactic-co-glycolic acid) (PLEA)conjugated with polyethylene glycol (PEG) (PEG-PI-GA) (5050 DLG, mPEG5000, 5 wt % PEG) was purchased from Lakeshore Biomaterials (Birmingham,Ala.). Loteprednol etabonate was purchased from Selleckchem (Houston,Tex.). Poly(lactide-coglycolide)-b-Poly(ethyleneglycol)-b-Poly(lactide-coglycolide) [PLGA-PEG-PLGA (Mn˜1,100:1,000:1,100Da, 3:1 LA:GA) (25% PEG)] thermogelling polymer was purchased fromPolyscitech (West Lafayette, Ind.). Phosphate buffered saline (PBS)solution was purchased from Mediatech, Inc (Manassas, Va.). Thiazolylblue tetrazolium bromide (MTT reagent), acetone and methanol werepurchased from Sigma Aldrich (St. Louis, Mo.). All other chemicals usedin the embodiment were of analytical grade and were used without anyfurther purification unless specified.

Loteprednol etabonate-loaded NPs (LIE NPs) were prepared using apreviously reported nanoprecipitation method. 8 mg of LE and 55 mg ofPEGylated PLEA were dissolved in 4 mL acetone (organic phase) and addeddropwise to 8 mL of deionized H₂O spinning at 300 rpm overnight to allowfull evaporation of the organic phase and the formation of the NPsuspension. The NPs were separated by centrifugation at 3,000 rpm for 10minutes and resuspended in fresh deionized H₂O.

Parameters of the NPs were studied by measuring the particle size andpolydispersity, index (PDI). The effect of loading LE into the NPs onthese parameters was studied by comparing LIE NPs to blank NPs. Particlesize and PDI were measured using the Dynamic Pro plate reader (WyattTechnology Corporation, Santa Barbara, Calif.). The NPs samples werediluted 1:5 in deionized water to fit instrument specifications.

Drug entrapment efficiency was determined by using a previously reportedmethod (20). 1 mL of the NPs solution was centrifuged at 12,000 rpm for5 minutes. The supernatant was removed and was replaced with 1 mL ofmethanol and stored at 4° C. overnight. The supernatant of theNPs/methanol solution was diluted 1:5 and measured by UV spectroscopy ata wavelength of 243 nm (λmax). The supernatant was analyzed and comparedto a series of standard dilutions of LE in methanol (r²=0.9981).Encapsulation efficiency was determined using the following equation:

${\%\mspace{14mu}{Entrapment}{\mspace{11mu}\;}{efficiency}} = {\frac{{Actual}{\mspace{11mu}\;}{drug}\mspace{14mu}{amount}}{{Theoretical}\mspace{14mu}{drug}\mspace{14mu}{amount}} \times 100}$

SEM was utilized to visualize the surface and physical integrity of LENPs. JOEL JSM-6490LV (JOEL Industries, Tokyo, Japan) was used tovisualize the samples. The samples were diluted according toinstrumental specifications and loaded onto aluminum cylinders coatedwith an adhesive carbon polymer. NP formulations were viewed in 15,000×magnification and 5 kv acceleration voltage was used to visualize thedrug loaded NPs.

TR gels were prepared using the cold method. Briefly, the 20% w/v gelwas prepared by solubilizing 100 mg of PLGA-PEG-PLGA gel in deionizedH2O overnight at 4° C. LE release rate from the NPs was measured by apreviously reported method. Dialysis membrane tubing, MWCO 10,000 Da.(Spectrum Laboratories, Rancho Dominguez, Calif.), was soaked indeionized H₂O overnight. NPs solutions were sonicated, and 1 mL wasadded to the dialysis membrane tubing; tubing was placed into 100 mL ofPBS and stirred at 100 rpm at 37° C. 1 mL samples were removed atpredetermined intervals over 7 days and replaced with fresh PBS. Sampleswere analyzed using UV spectroscopy at a wavelength of 243 nm (λmax) andcompared to standard dilutions of LE in PBS (r²=0.9915) to determine thepercentage of drug released over 10 days.

LE release from the TR gel was measured by allowing 200 μL of LE NPs in20% w/v polymer solution to gel by placing in incubator at 37° C. for 5minutes. After gelling, release was initiated by adding 2.5 mL PBS. 1 mLsamples were removed at predetermined intervals over 7 days and replacedwith fresh PBS.

Human retinal pigment epithelium, ARPE-19, cells (ATCC # CRL-2302) weregrown in 1:1 Dulbecco's Modified Eagle's Medium and Ham's F12 Medium(Mediatech, Inc., Manassas, Va.) with 10% fetal bovine serum and 100U/mL penicillin/streptomycin (Sigma Aldrich, St. Louis, Mo.). Thecultures were maintained in a humid environment at 5% CO₂ and 37° C.

Cytotoxicity was assessed in ARPE-19 cells by the3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide salt (MTT)assay. Briefly, cells were seeded in 24-well plates at a density of5×104 cells/mL for 24 hours to achieve confluence before treatment.Cells were exposed to free LE solution, LE NPs, and LE NPs-TR gel atvarying concentrations (0, 1, 10, 100 μM) for 24 hours after which theMTT assay was performed. Cell culture media were aspirated and 300 μL ofMTT reagent solution (1 mg/mL) was added to each well. Cells wereincubated at 37° C. and 5% CO₂ for 4 hours, after which the MTT reagentsolution was aspirated and 500 μL of DMSO was added to each well. Plateswere allowed to shake gently for 10 minutes before being read by SynergyH4 plate reader (Biotek Industries, Inc., Winooski, Vt.) at absorbanceof 570 nm.

ARPE-19 Cells Coumarin-6-loaded NPs were prepared by the method outlinedabove. In lieu of LE, 15 μL of coumarin-6 (1 mg/ml acetone stocksolution) was added. ARPE-19 cells were seeded at 10,000 cells per wellin a four-well chamber slide. The cells were incubated for 24 hours toachieve 70% confluence. The medium was aspirated and cell monolayer waswashed three times with PBS. 500 μL media containing 50 μg ofcoumarin-loaded NPs was added to the wells and incubated for 2 hours.After 2 hours, cells were washed with PBS three times, and treated withDAPI for nuclear staining and Cell Mask Deep Red for membrane staining.The slide was then examined by confocal microscopy with a magnificationof 60×.

ARPE-19 cells were seeded onto 24-well plates at 5×104 cells/mL andallowed to grow until confluence. On the day of study, cell culturemedia were replaced with 1% FBS experimental media and allowed to remainin quiescence for 12 hours. The cellular monolayers were incubated withvarying treatment concentrations of free LE solution (1, 10 μM), LE NPs(10 μM), and LE NPs-TR gel (10 μM). Culture media were collected at 12and 72 hours. Secreted VEGF in collected culture media was quantified bythe ELISA method (Human VEGFA ELISA kit, Thermo Scientific, Waltham,Mass.). The cell protein content was assayed using the BCA protein assaykit after lysing the cells and VEGF secretion was normalized to totalprotein. Samples were read by using the Synergy H4 plate reader (BiotekIndustries, Inc., Winooski, Vt.) with absorbance at 450 nm minusabsorbance at 550 nm.

Statistical analyses were performed using GraphPad Prism (GraphPadSoftware, Inc., San Diego, Calif.). Comparisons of the effect of free LEsolution, LE NPs, and LE NPs-TR gels on cell viability and VEGFexpression were assessed using paired t-tests with a significance level(p value) of 0.05. All experiments were carried out in triplicates (n=3)and shown as mean values±SD.

Dynamic Pro plate reader was used to determine the size and PDI of theblank and LE-loaded NPs. The LE NPs were larger in size than the blankNPs. PDI data revealed that all NPs formulations had a normal sizedistribution (Table 1) with PDI value less than 1.

NP Type Particle size (nm) PDI Blank NPs 125.10 ± 43.89 0.014 ± 0.004 LENPs 168.00 ± 23.18 0.0142 ± 0.0023

UV spectroscopy was used to determine the entrapment efficiency of LE bycomparing the absorbance in methanol to standard dilutions of LE inmethanol (r²=0.9981) at 243 nm (λmax). The LE NPs formulation showed anentrapment efficiency of 82.6±0.01% which was satisfactory.

SEM was used to visualize the morphology of LE NPs. Surface analysis ofNPs formulations showed that physical integrity was maintained by allsamples. A SEM visualization of loteprednol etabonate-loadednanoparticles shows round morphology. Samples were diluted 1:10 andvisualized by SEM. Samples were read at 15,000× magnification and 5 kVacceleration voltage, in accordance with an embodiment of the presentinvention. Size and size distribution of the NPs formulation visualizedthrough SEM corroborated with data obtained by DLS.

The release of LE from NPs and TR gel was conducted in vitro in a beakerwith 100 mL of PBS at 37° C. FIG. 6 shows the cumulative release profileof the Lot NPs formulation compared to the LE NPs TR gel. At 72 hours,the LE NPs formulation had released 88% of the encapsulated drug whilethe LE NPs TR gel formulation had released 5% of drug. At 168 hours, theTR gel had released 10% of drug.

The cytotoxicity of the DDS was investigated by MTT assay in the ARPE-19cells to determine any possible harm related to its use. FIG. 6 comparesthe cytotoxic effects of free LE with equal concentrations of the LE NPsand LE NPs in 20% w/v TR gel. FIG. 7 is a graphical illustration of MTTcytotoxicity data in ARPE-19 cells of increasing concentrations ofloteprednol etabonate (LE) free drug, LE NPs, and LE NPs in 20% w/v TRgel as compared to untreated control cells. Experiments were carried outin triplicates (n=3) and quantified by absorbance reading at 570 nm.Data is represented as mean±SD. Free LE had no impact on cell viabilityin ARPE-19 cells as compared to the untreated control cells. All othertreatments were not significantly cytotoxic in ARPE-19 cells.

The effect of the LE DDS on VEGF secretion was studied in ARPE-19 cells.Cells were treated with different concentrations of free LE solution, LENPs, and LE NPs in 20% w/v TR gel, for 12 and 72 hours. FIG. 8 comparesthe suppression of VEGF expression between free LE solution, LE NPs andthe LE NPs TR gel at 12 hours. Free LE solution at 1 μM and 10 μM showedreduction in VEGF expression compared to media control. LE NP at 10 μMwas used for the VEGF expression study due to slower releasecharacteristic of the NPs. Moreover, the preliminary data have shownbetter VEGF expression reduction at this concentration. LE NPs at 10 μMslightly reduced VEGF secretion while NPs TR Gel at 10 μM did not reduceVEGF secretion significantly in 12 hours compared to the control (pvalue <0.05) as concluded by paired t-test. However, the paired t-testresults indicated that both NPs and NPs TR Gel at 10 μM significantlyreduced VEGF secretion in 72 hours (p value <0.0), as shown in FIG. 9.This may be due to controlled release of the drug from NPs polymermatrix and NPs TR Gel. The VEGF expression for NPs and NPs TR Gel at 10μM was reduced to 90.99±1.29% and 96.24±1.91% respectively. The %reduction VEGF expression of NPs TR Gel was observed as lower than NPsdue to further delayed release of the drug from the gel matrix comparedto NPs alone.

Polymeric LE NPs were prepared by the emulsion solvent evaporationmethod with reproducible and satisfactory particle size and entrapmentefficiency. The particle size of unloaded NPs was 125 nm while drugloaded NPs was 168 nm. Both NPs formulations showed a normal sizedistribution with PDI value less than 1. Particle size, PDI, andentrapment efficiency data were corroborated with previously publishedstudies. The rate of drug release from the NPs and TR gel was measuredusing dialysis method in PBS at 37° C. over the course of 7 days. At 72hours, the LE NPs exhibited 88% drug release while the LE NP Gel hadreleased 5% of drug. The TR gel had released 10% of drug at 168 hours.Both formulations exhibited sustained release pattern; however, the drugrelease from the TR gel was further slower due to drug release viapolymer degradation. Polymer matrices release encapsulated drug in twophases: the first phase of drug release occurs via diffusion and thesecond phase is by diffusion and polymer degradation. MTT cytotoxicitystudies in ARPE-19 cells showed no cytotoxicity after 24-hour exposureto free LE solution, LE NPs, or LE TR gel. LE was not expected to haveany toxic effects as it was formulated to prevent any physiological sideeffects. The LE NP and LE NP incorporated TR gel DDS were observed to besafe to use in ARPE-19 cells. Additionally, the NPs were localized intothe ARPE-19 cells within 2 hours. To support these data, recent in vivostudies have shown that NPs were localized within 6 hours at the retinalpigment epithelium of healthy rat after intravitreal injections. LE NPsand LE NP TR gel were able to significantly reduce VEGF expression inARPE-19 cells at 10 μM over 72 hours more effectively than equalconcentrations of LE drug solution (p<0.05). Free LE drug solutionsignificantly reduced VEGF expression within 12 hours compared tocontrol (p<0.05) while drug loaded NPs and NPs TR Gel exhibit theireffect after 72 hours due to the extended release properties of the NPsand the gel.

This embodiment of the present invention provides for the use of a novelDDS for controlled release of LE for potential treatment of wet AMD byreducing the frequency of intravitreal injections. The proposed DDS ofthis embodiment showed sustained in vitro release and showed nocytotoxicity in in vitro experiments using ARPE-19 cells. The DDS of LEwas able to significantly reduce the expression of VEGF in ARPE-19 cellsover 72 hours compared to LE solution alone. The biocompatibility, lowtoxicity and therapeutic potential of the proposed DDS make it an idealmodel treating AMD and reducing intravitreal injection frequency.

It will be seen that the advantages set forth above, and those madeapparent from the foregoing description, are efficiently attained andsince certain changes may be made in the above construction withoutdeparting from the scope of the invention, it is intended that allmatters contained in the foregoing description or shown in theaccompanying drawings shall be interpreted as illustrative and not in alimiting sense.

It is also to be understood that the following claims are intended tocover all of the generic and specific features of the invention hereindescribed, and all statements of the scope of the invention which, as amatter of language, might be said to fall therebetween.

What is claimed is:
 1. A drug delivery system for the treatment ofage-related macular degeneration (AMD), the drug delivery systemcomprising: a sustained-release formulation comprising; a PLGA-PEG-PLGAthermoreversible (TR) hydrogel; and PLGA nanoparticles encapsulatingloteprednol etabonate (LE) incorporated into the PLGA-PEG-PLGAthermoreversible (TR) hydrogel, wherein the PLGA nanoparticles have anentrapment efficiency of LE equal to 82.6±0.01% and wherein the PLGAnanoparticles have an average particle size equal to 168.00±23.18 nm;wherein the sustained-release formulation provides a burst-free,sustained release of the TA during both a diffusion phase and a polymerdegradation phase of the sustained release.
 2. The drug delivery systemof claim 1, wherein the PLGA-PEG-PLGA TR hydrogel is a 20% w/v TRhydrogel.
 3. The drug delivery system of claim 1, wherein the PLGAnanoparticles are substantially spherical.
 4. A method for the treatmentfor age-related macular degeneration (AMD), the method comprising:administering one or more intravitreal injections of a sustained-releaseformulation comprising PLGA nanoparticles encapsulating loteprednoletabonate (LE) incorporated into a PLGA-PEG-PLGA thermoreversible (TR)hydrogel to a patient, wherein the sustained-release formulationprovides a burst-free, sustained release of the LE during both adiffusion phase and a polymer degradation phase of the sustained releaseand wherein the PLGA nanoparticles have an entrapment efficiency of LEequal to 82.6±0.01% and wherein the PLGA nanoparticles have an averageparticle size equal to 168.00±23.18 nm.
 5. The method of claim 4,wherein the PLGA-PEG-PLGA TR hydrogel is a 20% w/v TR hydrogel.
 6. Amethod of manufacturing a drug delivery system for the treatment ofage-related macular degeneration (AMD), the method comprising; formingPLGA nanoparticles encapsulating a loteprednol etabonate (LE); andincorporating the PLGA nanoparticles encapsulating LE into aPLGA-PEG-PLGA thermoreversible (TR) hydrogel to from a sustained-releaseformulation, wherein the sustained-release formulation provides aburst-free, sustained release of the LE during both a diffusion phaseand a polymer degradation phase of the sustained release and wherein thePLGA nanoparticles have an entrapment efficiency equal to 82.6±0.01% andwherein the PLGA nanoparticles have an average particle size equal to168.00±23.18 nm.
 7. The method of claim 6, wherein the PLGA-PEG-PLGA TRhydrogel is a 20% w/v TR hydrogel.
 8. The method of claim 6, wherein theloteprednol etabonate encapsulated nanoparticles are substantiallyspherical.